2.4 Doença Inflamatória Intestinal
3.7.3 Avaliação do Dano Intestinal
3.7.3.7 Análise Imunohistoquímica de COX-2 e iNOS
Três cortes finos (4 μm) de cólons (3 animais por grupo) foram obtidos com um micrótomo e transferidas para lâminas lâminas silinizadas. Cada corte foi desparafinizado e reidratado. Secções de tecido do cólon foram lavadas com 0,3% de Triton X-100 em tampão fosfato, colocado peroxidase endógena (3% de peróxido de hidrogénio), e incubadas durante a noite a 4 °C com os seguintes anticorpos primários: COX-2, 1:600; iNOS, 1:700 (Santa Cruz, USA). Depois, as seccções foram lavadas com tampão fosfato e incubadas com um anticorpo secundário estreptavidina-HRP-conjugada (Biocare Medical, Concord, CA, EUA) durante 30 minutos, e a imunorreactividade de COX-2 e iNOS foi visualizada utilizando um kit de detecção baseada em colorimétrico seguindo o protocolo fornecido pelo fabricante (TrekAvidin-HRP Label + Kit from Biocare Medical, Dako, USA). Controles positivos e negativos foram incluídas em cada grupo. Foi utilizado microscopia planimétrica (Olympus BX50, Departamento de Morfologia/UFRN) com objetiva de alta potência (40x). A intensidade da imunocoloração de células foi determinada e score de 1 a 4 foi atribuído: 1= ausência de células positivas; 2 = pequeno número de células positivas ou células isoladas; 3 = número moderado de células positivas; e 4 = elevado número de células positivas. A intensidade de marcação foi avaliada por dois examinadores previamente treinados, de forma duplo-cego. Foram avaliados três cortes por animal.
3.8 Análise Estatística
As análises das diferenças entre as médias foram avaliadas através da análise de variância de uma via (ANOVA), seguido do teste de Tukey. Os dados não paramétricos (escore) foram analisados utilizando-se o teste de Mann-Whitney. O nível de significância foi estabelecido em p <0,05, utilizando o programa estatístico GraphPad Prism 4.
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APÊNDICE A
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